Time signals of expression or modification of a protein engaged in proliferation, differentiation or development of various cells are now being clarified. When applying these findings to engineering fields such as regeneration medicine or tissue engineering, if it is possible to control the existence of a protein intended to function in cells at a designated amount for a designated period, it is expected to broaden various potentialities.
As a technique to enable a designated protein to function in cells, gene transfer is almost the only method available at present. When constant functioning of the designated protein in cells is desired, gene transfer is effective. However, when the designated protein is desired to transiently function in cells, it is preferable to transduce the protein itself into the cells.
Conventionally, as a method for transducing a protein itself into cells, because of the necessity of traverse through cell membranes, examples of methods to be used include a special method such as microinjection, and a method wherein a protein is encapsulated into a capsule-shaped material such as a liposome and the capsule shaped-material is fused with the cell membrane so as to transduce the content (protein etc.) of the capsule-shaped material into the cell. In addition, though the kinds of cells are limited, another method for transducing a protein into cells through a receptor-dependent route has come into practical use, wherein various receptors expressed on cell surfaces are targeted and ligands of these receptors are used as carriers.
The inventors have recently confirmed that apart from these routes, a highly cationic protein or a protein cationized by chemical modification was electrostatically adsorbed to a negatively charged cell surface, and accumulated in cells with high efficiency (Futami et al., Biochemistry, 40, 7518-7524, 2001). Likewise, recently it has been reported that a protein to which a highly basic TAT peptide derived from HIV (Schwarze et al., Science, 285, 1569-1572, 1999) or a cationic peptide (Futaki et al., J. Biol. Chem., 276, 5836-5840, 2001) such as Poly-Arg is added, can efficiently cross a cell membrane. Although in all the cases mentioned above, mechanisms have yet to be known in detail, a cell membrane crossing route mediated by the electrostatic interaction between a cationic protein and a cell surface is supposed.
However, a conventional protein cationization method requires modification of many amino acid side chains in a protein molecule, thereby deteriorating its function.